Studies On The Genomic Instability Of Bone Marrow Mesenchymal Stem Celts Induced By Paclitaxel

Background:Along with the progress of the society, the environment, and the changeof lifestyle，Malignant tumor has become a threat to human health and life. the morbidity andmortality of it has increased year by year. Therefore, early detection and early diagnosis ofthe malignant tumor is necessary.There are many different theory about the occurrence of malignant tumor, manyscholars have focused on the theory of cancer stem cells. The theory believe that the tumor isa kind of diseases of stem cells.Imbalance of normal self-renewal and differentiation willhappen when the genomic instability of the stem cells happen, Stem cells will gradually turninto cancer stem cells, and then form tumor. Genomic instability is a key events in the earlyperiod of tumor forming, The physical and chemical factors,such as Ionizing radiation,chemical agent can cause genetic instability.Genomic instability include four types,regional chromosomal instability with high frequencies of occurrence is the most importantone.This experiment aim at regional instability of chromosome，we established a Greenfluorescent protein（GFP）labeled genomic instability reporting system（The plasmidpatterning on chapter3materials and methods, and figure3.1）, using repeat GFP as areporter factor, due to a gene XhoI inserted which makes the GFP inactivation. Whenhomologous recombination occurred between the two repeat GFP, GFP-positive cells will beproduced,and the cells express green fluorescent. In addition，the reporting system encodesNeor gene,which is a neomycin resistance gene.The cells will receive G418resistence andgrow in selective medium with G418after the system established. Thus we get cells labeledwith GFP to establish genomic instability reporting system by G418selection.Objective:To test the genomic instability of bone marrow mesenchymal stemcells(BMSCs）induced by paclitaxel, using GFP labeled genomic instability reportingsystem.Method:Used the electroporation transfection, the genome instability report systemmarked by GFP will be established in BMSCs. After transfection, G418screening,limitingdilution. The BMSCs with the reporter plasmid were dealed with different doses of paclitaxel (5μM，10μM，15μM，20μM，30μM), and genomic instability of BMSCs inducedby paclitaxel was quantified detected by flow cytometry at4h、24h、48h and4h+96h afterpaclitaxel introduction.Results:The results suggest that:1.The GFP marked regional chromosomal instabilityreporter had transfected BMSCs successfully.2.The final concentration of G418selection ofBMSCs lines was100μg/ml, and the maintain concentration was50μg/ml.3. GFP’sexpression happen at4h, and was time-dependent. GFP expression rate：4h＋96h＞48h＞24h＞4h.4.GFP expression will happen at the dose of5μM, different doses of paclitaxel（5μM、10μM、15μM、20μM、30μM）could induce different expression of GFP, GFP’sexpression was positively correlated with paclitaxel dose, GFP expression rate：30μM＞20μM＞15μM＞10μM＞5μM.Conclusions:The results suggest that:1.The GFP marked regional chromosomalinstability reporter had transfected BMSCs successfully.2.BMSCs can be successfullyinduced regional chromosomal instability by paclitaxel and can be detected by this GFPreporting system.3. Different doses of paclitaxel could induce different expression of GFP,GFP’s expression was increased with the increase of the concentration of the paclitaxel andfunction the extension of time.4. paclitaxel-induced regional chromosomal instabilitychanges can be passed to offspring cells.